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Figure 6


Fig. 6. A small intracellular domain of Lrp6 is sufficient to mediate its convergent-extension activity. (A) Serial deletions of the intracellular domain of Lrp6 demonstrate that a 36 amino acid fragment, Lrp6-B, can mediate its convergent-extension activity. Dark red boxes indicate PPP(S/T)P motifs. All constructs have an N-terminal myristoylation sequence. Percentages of normal (>70% ACEL; average control embryo length), mildly affected (30-70% ACEL), or severely affected (<30% ACEL) embryos are shown (bar graph). Each construct (500 pg mRNA) was used in at least three independent experiments. Numbers of embryos scored are shown in parentheses. (B) Sequence comparison of the B domain of human Lrp6 (hLRP6-B), mouse Lrp6 (mLRP6-B) and Xenopus Lrp6 (XLRP6-B). Numbers in parentheses indicate the amino acid position of the start of the B domain. Identical amino acids are in blue. (C) Embryos and explants from embryos injected with lrp6-B mRNA (2 ng) show severe convergent-extension defects; Lrp6-B (1.6 ng) synergizes with Lrp6 (1 ng) in blocking animal cap elongation. Lateral views of stage 26 embryos are shown. In the bar graph, single asterisks indicate statistically significant differences from control (P<0.01), and double asterisks indicate statistically significant differences from Lrp6 and Lrp6-B values (P<0.01). Numbers of caps scored are indicated in parentheses. (D) Lrp6-B does not induce expression of Wnt/ß-catenin target genes, siamois and nr3 (Xnr3), or act in a dominant-negative manner to inhibit Wnt8-induced expression of siamois and nr3 in animal caps. Loading control: ODC (ornithine decarboxylase). Whole embryos (WE) were used as positive control. RT, reverse transcriptase. (E) In contrast to dominant negative Dishevelled (Xdd), transient transfection of Lrp6-B in HEK293 cells does not upregulate TOPFLASH or inhibit Wnt3a-induced TOPFLASH activity. Student's t-test was used for statistical analysis. Error bars indicate standard deviation. Scale bar: 500 µm in C.





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